1. Field of the Invention
This invention relates to a composition for ethanol assay.
2. Description
The testing of body fluids, such as saliva, blood, and urine, for determining the amount of ethanol therein is important for safety and health reasons, among others. Abuse of alcohol and detection thereof together with determination of blood alcohol content when a person is suspected of driving while intoxicated are two of many important areas where quick and reliable ethanol determination is important.
A useful method of determining the ethanol content of body fluids utilizes the enzymatic reaction whereby ethanol is converted to acetaldehyde by the action of alcohol dehydrogenase (ADH) and where nicotinamide adenine dinucleotide (NAD), acting as a coenzyme, is converted to its reduced form (NADH), as shown in the reaction sequence below: ##STR1##
The equilibrium of this reaction lies on the side of ethanol and NAD. However, the equilibrium is displaced to the right when the reaction takes place under alkaline conditions and the acetaldehyde formed is trapped. The course and extent of the reaction can be determined by spectrophotometric resolution at 340 nanometers. At this wavelength, NAD does not absorb ultraviolet light, but NADH does absorb ultraviolet light. The amount of NADH formed in the above reaction corresponds to the amount of ethanol present.
To be used as an assay, the reaction must go to completion. However, because of the unfavorable equilibrium of the reaction when ethanol is oxidized to acetaldehyde by NAD, the acetaldehyde has to be removed from the system so that NADH is quantitatively formed with respect to the ethanol. One way to remove, or trap, the acetaldehyde is to use a trapping agent. Additionally, as with most enzymatic reactions, a buffer is necessary to maintain the pH of the assay system within the optimum range for the particular enzymatic reaction.
It is also known that trapping agents presently used can inactivate ADH. Current assay systems have low stability once mixed, less than one day at room temperature (about 20.degree. C.) and about 3 days if refrigerated. Fresh assay reagents must be prepared frequently and low stability may lead to a more frequent need to run known ethanol samples to ensure accuracy.
Many traping agents currently used in ethanol assays and reagents, for example, hydrazine, can inactivate ADH, limiting the shelf stability of the assay or reagent.
Thus, there exists the need for a trapping agent that does not inactivate ADH. There also exists need for an enzyme stabilizing composition for the ADH. There also exists the need for an assay system which has long stability.